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Old 09-27-2019, 02:52 PM   #1
Carcharodon
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Location: Honolulu, HI

Join Date: Jul 2015
Posts: 40
Default Is this library overamplification? Is this problematic?

Aloha all,

I definitely DO have library overamplification in some of my libraries (as evidenced by a secondary peak, likely the result of the "daisy-chaining" effect). But that's not really what has me curious.

I've attached an image below of the electropherogram of two of my libraries. What has my attention is the strong peak around 400 bp (~ 407 bp), and the small shoulder to the left (~ 380 bp), especially in D702. D709 has both peaks, but there is more "equal" representation of the sizes/fragments across the range. Libraries like D702 are in the majority.

After Pippin-Prep size selection, the elution was split into four separate PCRs, then pooled and cleaned. So this pattern has emerged independently not only across libraries, but presumably across independent PCRs within libraries, with remarkable consistency.

My question is: is this the result of overamplification (my original size-selection encompassed a range of ~ 70 bp, so it's tight), and could this reduce the complexity of my libraries? Or is this simply what I might expect to see given a relatively tight size-selection?

Side-note: I'm using such a tight size-range because my study organism has a 7-8 Gbp genome.




Last edited by Carcharodon; 09-27-2019 at 03:02 PM.
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